Rat and chicken s-rex/NSP mRNA: nucleotide sequence of main transcripts and expression of splice variants in rat tissues

Abstract

Two main transcripts of the s-rex/NSP gene are generated by different promoter usage and differential splicing in neuronal and endocrine tissues of higher vertebrates, suggesting that the encoded proteins function in neuroendocrine secretion. To know more about the structure, expression and evolution of this new gene, we have cloned full-length cDNAs for both 1.5 kb and 3.5 kb transcripts from rat and chicken brain cDNA libraries. Sequence analysis has revealed structures within the 3′-UTR that are conserved in these mRNAs and human NSP mRNA and that could be involved in specific compartmentalization of s-rex/NSP mRNA in neuronal cells. An additional transcript generated by differential splicing of internal exons has been cloned from a rat DRG library. Low levels of s-rex/NSP mRNAs have been detected in some non-neuroendocrine tissues, although substantial levels of a unique transcript have been found in rat testis. By RT-PCR analysis, other tissue-specific transcripts that are products of rare splicing events have been revealed.

Introduction

Compartmentalization of mRNA is a mechanism used by different types of eukaryotic cells to regulate the specific spatial distribution of encoded proteins (reviewed by Ding and Lipshitz, 1993; Wilhelm and Vale, 1993; St Johnston, 1995). This mechanism is especially important in case of proteins lacking specific localization signals and for large polarized cells. It has been suggested that in neurons, localization of specific mRNA followed by protein synthesis in postsynaptic sites could be important for nervous system development and neuronal plasticity (Craig and Banker, 1994; Steward, 1995). To clone cDNA for transcripts with a predominantly synaptic localization we used a subtractive cloning technique (Baka and Buchman, 1996). Clones for mRNA that are more abundant in synaptosomal than in cytoplasmic fractions of juvenile rat cerebral cortex have been isolated. One of these clones, named s-rex, had a high degree of homology to the human NSP gene, the first member of the reticulon family, isolated from a SCLC cell line (Roebroek et al., 1993). Two NSP transcripts (1.5 kb and 3.5 kb) are expressed in human neuronal and endocrine tissues. Using human NSP and rat s-rex cDNA as hybridization probes, two transcripts with sizes similar to human NSP transcripts have been detected in the CNS and PNS of embryonic and newborn rats (van de Velde et al., 1994a) and chicken (Baka et al., 1996). The encoded proteins are not analogous with proteins with known functions. They possess two hydrophobic domains and were localized on membranes of the endoplasmic reticulum (van de Velde et al., 1994b). The exact function of s-rex/NSP is not clear but structural, expression and localization studies suggest the involvement of these proteins in neuroendocrine secretion. Here we report the cloning and nt sequence of a full-length cDNA for the two main rat and chicken s-rex transcripts, a rare third type of s-rex transcript from rat DRG and detection by Northern hybridization and RT-PCR of other types of transcripts in rat tissues.